Triorthogonal reagents for dual protein conjugation

Patent metadata

Field	Value
Publication number	US-9878045-B2
Application number	US-201415029599-A
Country	US
Kind code	B2
Filing date	Oct 15, 2014
Priority date	Oct 15, 2013
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The present invention relates to triorthogonal reagents useful for site-specifically modifying a protein with two orthogonal groups that can be subsequently functionalized in a single one-pot procedure. This approach relies on the selective tagging of proteins containing an appended farnesyltransferase or geranylgeranyltransferase I substrate sequence. The incorporation of a bifunctional ethynyl-hydroxybenzaldehyde into the farnesyl or geranylgeranyl group facilitates the facile labeling of proteins with two different moieties.

First claim

We claim:

1. A compound of formula I or a salt thereof:

wherein n is an integer from 1 to 2.

2. A compound of formula II or a salt thereof:

wherein
n is an integer from 1 to 2; and
R 1 is a protein containing a CaaX motif which is attached the cysteine residue of said CaaX motif.

3. The compound of claim 2 , wherein the CaaX motif is the amino acid sequence CVIA (SEQ ID NO: 1).

4. The compound of claim 2 , wherein R 1 is Ciliary Neurotrophic Factor.

5. A compound of formula III or a salt thereof:

wherein:
n is an integer from 1 to 2;
R 1 is a protein containing a CaaX motif which is attached the cysteine residue of said CaaX motif;
X is O or NH;
F 1 ′ is a first functional group; and
F 2 ′ is a second functional group.

6. The compound of claim 5 , wherein the CaaX motif is CVIA (SEQ ID NO: 1).

7. The compound of claim 5 , wherein X is O.

8. The compound of claim 5 , wherein X is NH.

9. The compound of claim 5 , wherein F 1 ′ is a first functional group formed from reacting a compound of formula II with a compound F 1 containing an azide group that reacts with the ethynyl group of the compound of formula II to form a triazole linkage thereto.

10. The compound of claim 5 , wherein F 2 ′ is a second functional group formed from reacting a compound of formula II with a compound F2 containing an aminooxy group that reacts with the formyl group of the compound of formula II to form an oxime linkage thereto.

11. The compound of claim 5 , wherein F 2 ′ is a second functional group formed from reacting a compound of formula II with a compound F2 containing an hydrazinyl group that reacts with the formyl group of the compound of formula II to form a hydrazone linkage thereto.

12. A method of functionalizing a protein having a CaaX motif, comprising:
(a) reacting said protein with a compound of formula I or a salt thereof in the presence of a protein farnesyltransferase or a geranyl-geranyltransferase I to produce a compound of formula II or a salt thereof:

wherein
n is an integer from 1 to 2; and R 1 is said protein which is attached to the remainder of the compound at the cysteine residue of the CaaX motif; and
(b) reacting the compound of formula II or a salt thereof with a first functional compound F 1 containing an azide group that reacts with the ethynyl group of the compound of formula II to form a triazole linkage thereto, and a second functional compound F 2 containing a reactive aminooxy or hydrazine group that reacts with the formyl group of the compound of formula II to form an oxime or hydrazone linkage thereto, thereby forming a compound of formula III or a salt thereof:

wherein
X is O or NH;
F 1 ′ is a first functional group; and
F 2 ′ is a second functional group.

13. The method of claim 12 , wherein n is 1.

14. The method of claim 13 , wherein the compound of formula I is reacted with said protein in the presence of protein farnesyltransferase.

15. The method of claim 12 , wherein n is 2.

16. The method of claim 15 , wherein the compound of formula I or a salt thereof is reacted with said protein in the presence of geranylgeranyltransferase I.

17. The method of claim 12 , wherein the CaaX is CVIA (SEQ ID NO: 1).

18. The method of claim 12 , wherein R 1 is Ciliary Neurotrophic Factor.

19. The method of claim 12 , wherein the first functional compound F 1 is azido-TAMRA fluorophore.

20. The method of claim 12 , wherein the first functional compound F 1 is azido-bis-methotrexate.

21. The method of claim 12 , wherein the second functional compound F 2 is aminooxy-PEG.

22. The method of claim 12 , wherein the second functional compound F 2 is aminooxy-TAMRA.

23. The method of claim 12 , wherein the first and second functional compounds F 1 and F 2 are reacted with the compound of formula II simultaneously.

24. A pharmaceutical composition comprising a compound of formula III or a salt thereof as described in claim 12 and a pharmaceutically acceptable carrier.

25. A method of functionalizing a protein having a CaaX motif, comprising:
a) reacting said protein with a compound of formula I or a salt thereof:

wherein X and Y are bioorthogonal groups that are identical or different which are capable of conjugating to a functional compound; Z is H, OH, halogen or haloalkyl; and n is an integer from 1 to 2; in the presence of a protein farnesyltransferase or a geranyl-geranyltransferase I to produce a compound of formula II or a salt thereof:

wherein
X and Y are bioorthogonal groups that are identical or different which are capable of conjugating to a functional compound; Z is H, OH, halogen or haloalkyl; and n is an integer from 1 to 2; and R 1 is said protein which is attached to the remainder of the compound at the cysteine residue of the CaaX motif; and
(b) reacting the compound of formula II or a salt thereof with a first functional compound containing a reactive group that reacts with X to form a linkage to said first functional compound, and a second functional compound containing a reactive group that reacts with Y to form a linkage to said second functional compound, thereby forming a compound of formula III or a salt thereof:

wherein
F 1 ′ is a first functional group; and F 2 ′ is a second functional group.

Assignees

• Univ Minnesota

Inventors

• Distefano Mark
• Rashidian Mohammad

Classifications

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• A61K47/62 Primary

  the modifying agent being a protein, peptide or polyamino acid · CPC title

• A61K47/481 Primary

  Human Necessities · mapped topic

• A61K47/55

  the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug · CPC title

• C07F9/098 Primary

  Esters of polyphosphoric acids or anhydrides · CPC title